The specific aims of the proposed research are to characterize the molecular structure and function of the t complex responder gene (Tcr) in mouse t haplotypes. t haplotypes are variant forms of the proximal third of mouse chromosome 17, which encode multiple mutant loci affecting sperm differentiation. Tcr is the crucial locus in the t haplotype phenomenon of male transmission ratio distortion (TRD), a mechanism responsible for the propagation of t haplotypes through wild mouse populations. TRD involves at least four t haplotype-encoded "distorter" loci acting in concert with the responder to cause distortion (in +/t males) and sterility (in t/t males). Tcr is the only mammalian gene known to have a haploid effect on the outcome of sperm differentiation. In preliminary work, reverse genetics techniques were applied to clone a strong candidate for Tcr, called the T66 gene. To determine whether the T66 gene indeed represents Tcr, transgenic mice will be constructed, bred, and assayed for responder phenotypes. This approach should reveal general principles of haploid gene regulation. The T66 gene is a member of a multi-gene family. Genetic evidence for a second responder locus correlate strongly with other T66 gene family members. We will characterize the transcriptional activity of the various T66 gene homologues, their patterns of alternative message processing, and their intron-exon structures. This will be accomplished by performing primer extension analyses, S1 nuclease protection assays, and sequencing of genomic and cDNA clones. Chromosomal walking will be continued in order to complete a physical linkage of the DNA encompassing the responder locus. These studies will permit a characterization of the duplication events which have shaped the T66 family, and may provide clues to structure-function relationships. The long range goal of this work is to understand the molecular basis of TRD and associated sterility. Characterizing the Tcr gene will provide the foundation for future experiments designed to understand the responder/distorter interactions. The multifacted nature of the TRD system provides a unique resource for the study of spematogenesis, genome evolution and complex development systems in general.